Method of producing a bacteriological insecticide



United States Patent Ofiice 3,190,811 Patented June 22, 1965 3,190,811METHOD OF PRODUCING A BACTERIOLOGICAL INSECTICIDE Charles W. Molander,14816 Bora Drive, La Mirada, Calif. No Drawing. Filed June 14, 1963,Ser. No. 287,754 2 Claims. (Cl. 195-96) This is a continuation-in-partof application Serial No. 10,073, filed February 23, 1960, and nowabandoned.

This invention relates to a new biological insecticide produced by"certain bacteria in the absence of sporulation or spores and whichinhibits the development of insect larvae and pupae.

A great need exists forbacteriological insecticides for the control ofvarious insects, particularly in the dairy industry, where the fecalmaterial of animals is a major area for development of flies and otherinsects. Besides spraying and other conventional methods, it has beenpro-, posed to control the development of such insects by feedinganimals with food containing a suitable insecticidal substance so thatthe insecticidal substance will pass through the digestive tract of theanimal and will be present in the fecal material of the animals, therebyinhibiting the development of insect larvae or pupae present thereineither originally or by deposition by adult insects.

Such an insecticidal substance must retain its insecticidial activityasit passes through the digestive tract of animals and must also benon-toxic to the animals involved. Previous attempts to develop aneffective insecticide with the required qualities have been largelyunsuccessful so far as the dairy industry is concerned.

Other difiiculties in the past consisted in obtaining an effectivebacteriological insecticide in a relatively short time of production andin a yield sufficient for commercial use at a relatively low cost. Ihave been able to produce a suitable bacteriological insecticide insufficient amount for commercial use after a relatively short time ofincubation and in the absence of spores or sporulation of the bacteriainvolved.

It is, therefore, an object of my invention to provide a bacteriologicalinsecticide which inhibits the growth of insect larvae and pupae,particularly of the family Musciade in'the order Diptera.

Another object of my invention is to provide an effectivebacteriological insecticide which is produced .in a

relatively short time of incubation in the absence of bacterial sporesor bacterial sporulation and in commercially practicable amounts.

A further object of my invention is to provide a bacteriologicalinsecticide which is non-toxic to animals when fed to them in theirfood.

A still further object of my invention is to provide a bacteriologicalinsecticide which retains its insecticidal activity after passingthrough the digestive tract of animals.

These and other objects will be more readily understood by reference tothe following discussion and claims.

According to the present invention, the novel biological insecticide isproduced by fermentation of a quantity of Bacillus thuringiensis var.thuringz'ensis under controlled conditions in the absence of bacterialspores or sporulation. The novel insecticide may also be produced in thesame manner by using a quantity of the organism Bacillus thuringiensisvar. majumder.

The organisms B. thuringiensis var. thuringlensis and B. thuringiensisvar. majumder are commercially available at the present time. They maybe maintained on nutrient agar or other suitable media, and transferredabout once a month by standard bacteriological procedures.

To accomplish proper fermentation of the organisms to produce thebiological insecticide, a suitable nutrient media is used including anitrogen source and a carbohydrate source diluted in water. Vitaminsandother minerals may also be included.

The pH of the nutrient media may vary from 6 to 8, but the optimum pHfor maximum production of the biological insecticide is 6.8, which iscritical.

The temperature at which the nutrient media is maintained is alsocritical for optimum yields, and is 30 C. However, it is possible tovary the temperature from 20 C. to 40 C. and still obtain some amountsof insecticidal activity.

These conditions of time, temperature, and nutrient media are maintainedfor a maximum period of 16 hours. I have found that the biologicalinsecticide is produced in maximum yield in the absence of any spores ofsporulation of the bacteria.

Tests were made of the culture media to determine the absence of spores.or spor-ulation after incubation times of 6, l2, and 16 hours. Themethod used was that of Schaeffer and Fulton using a Malachite Greenstain for spores as modified by Ashby in Science 87,433 (1938). Theprocedure is as follows:

A sample of the culture media (containing both liquid and solidmaterial) is obtained with a loop and placed on a glass slide as a film.The film is dried and fixed on the glass slide 'with minimal flaming.The slide is placed over a beaker of boiling water, resting it on therim with the bacterial film uppermost. Within several seconds, largedroplets of water condense on the bottom side of the slide and the slideis then flooded with a 5% solution of Malachite Green which is allowedto act for one minute while the water continues to boil. Thereafter, theslide is washed in cold water and then flooded with 0.5% safranine for30 seconds and then washed, dried and examined microscopically forpresence of spores which are stained green under this method.

Under the above method for detection of spores, no spores or sporulationwere observed at incubation times of 6, 12, or even at the maximumincubation time of 16 hours of the culture media used to produce thenovel biological insecticide.

A typical example illustrating a preferred method of production of mynovel insecticidal substance follows.

Example 1 A culture media is prepared comprising by weight approximately3.0% of beet or other type of molasses, 0.6% yeast, 0.3l.5% corn steepliquor, 0.2% KH PO and 0.6% animal stick liquor. Other suitable aqueousculture media may also be used so long as a nitrogen source and acarbohydrate source are present. A quantity of Bacillus thuringienszsvar. thuringiensis is then inoculated into this culture media which isthen allowed to ferment at 30 C. at a pH of 6.8 under aerobic conditionsfor a maximum of 16 hours. At the end of 16 hours, sam les of theculture media were tested for the presence of spores or sporulation bythe method previously described. None was found. The culture media wasthen tested for insecticidal activity and was found to containsubstantial insecticidal activity.

The culture media was then centrifuged for 20 minutes at 4,000 rpm. andthe supernatant liquid was filtered through a Whatman No. l filterpaper. The filtrate was tested for insecticidal activity and was foundto contain approximately the same amount of activity as the originalculture media. Extraction of the filtrate bydiethyl ether did not removethe insecticidal activity.

Example 2 The same procedure was followed as in Example 1. After 16hours of incubation, no spores or sporulation was found. The resultingculture media was adjusted to a pH of 4.5 and extracted with diethylether. This extraction did not remove the insecticidal activity of theculture media. Then the culture media was adjusted to a pH of 4.5 andcentrifuged and filtered as before in Example 1. The filtrate containedapproximately the same insecticidal activity as the original culturemedia. Extraction of the filtrate by diethyl ether failed to remove theinsecticidal activity from the filtrate.

No detectable insecticidal activity is lost by drying the resultingculture media for one hour at 70 C. Redissolving the dried material inthe original volume of water produced no decrease in insecticidalactivity. However, boiling for minutes destroyed about 50% of theinsecticidal activity.

The filtrate containing the insecticidal activity may be applied in anyconventional manner such as spraying, etc. it may also be fed to animalsas part of their food. Experiments in animal feeding show that no toxiceffect was produced on the animals by the insecticidal substance. Aftersuch feeding, experiments showed that the developmerit of insect larvaeand pupae, particularly in the family Muscidae in the order Diptera,present in the fecal material of the animals receiving the insecticidalsubstance, was inhibited.

Although I have described preferred embodiments of my invention as tomethods of preparation and compositions of matter, it is understood thatthe scope of the invention is not to be limited thereby but numerousvariations are possible without departing from the spirit and scope ofthe invention and claimed hereinafter.

I claim:

1. A process for producing an insecticidal substance exercising aninhibiting action on insect larvae and pupae of the family Muscidae inthe order Diptera, comprising, fermenting the organism Bacillusthuringiensis var. tlzuringiensis under aerobic conditions in an aqueousnutritive media containing by weight approximately 3.0% beet molasses,0.6% yeast, 0.3-1.5% corn steep liquor, 0.2% KH PO and 0.6% animal stickliquor, at a pH of 6.8 at a temperature of 30 centigrade for a period ofup to 16 hours to prevent sporulation, centrifuging the resultingculture medium, and filtering the resulting supernatant liquidcontaining substantial insecticidal activity therein devoid of anyspores and devoid of sporulation.

2. The process of claim 1 wherein the organism fermented is Bacillusthuringiensis var. majumder.

References Cited by the Examiner UNITED STATES PATENTS 1/63 Bonnefoi..195% 1/63 Megna 96 OTHER REFERENCES A. LOUIS MONACELL, PrimaryExaminer.

1. A PROCESS FOR PRODUCING AN INSECTRICIDAL SUBSTANCE EXERCISING ANINHIBITING ACTION ON INSECT LARVAE AND PUPAE OF THE FAMILY MUSCIDAE INTHE ORDER DIPTERA, COMPRISING, FERMENTING THE ORGANISM BACILLUSTHURINGIENSIS VAR. THURINGIENSIS UNDE AEROBIC CONDITIONS IN AN AQUEOUSNUTRITIVE MEDIA CONTAINING BY WEIGHT APPROXIMATELY 3.0% BEET MOLASSES,0.6% YEAST, 0.3-1.5% CORN STEEP LIQUOUR, 0.2% KH2PO4, AND 0.6% ANIMALSTICK LIQUOR, AT A PH OF 6.8 AT A TEMPERATURE OF 30* CENTIGRADE FOR APERIOD OF UP TO 16 HOURS TO PREVENT SPORULATION, CENTRIFUGING THERESULTING CULTURE MEDIUM, AND FILTERING THE RESULTING SUPERNATANT LIQUIDCONTAINING SUBSTANTIAL INSECTICIDAL ACTIVITY THEREIN DEVOID OF ANYSPORES AND DEVOID OF SPORULATION.